Engineering of Sensory Proteins with New Ligand-Binding Capacities

Tavares, Diogo; Maffenbeier, Vitali; Meer, Jan Roelof van der

doi:10.1007/978-3-319-47405-2_129-1

Cited by 3 publications

(2 citation statements)

References 123 publications

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“…The function of many proteins in living organisms is initiated by the binding of specific molecules. Accordingly, a large panel of natural ligand-binding proteins, such as periplasmic binding proteins (PBPs) [4,5] and lectins, [6] has been exploited to constitute a molecular recognition unit in a sensor system, followed by combination with a reporter domain that converts a binding event into detectable signals or gene expression. [3,7] Among ligand-binding proteins, the PBP family is steadily employed as a scaffold for a binding module owing to its intrinsic features, such as its innate narrow specificity and affinity towards corresponding small molecules.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Q-SHINE: a versatile sensor for glutamine measurement via ligand-induced dimerization

Seo

Lim

Jung

et al. 2022

Preprint

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Biosensors for ligands are versatile tools for biological applications ranging from disease diagnosis to the detection of environmental chemicals. Thus, expanding the repertoire of biosensor toolkits provides novel insights into the unappreciated potentials of molecules. While the natural recognition domains have been employed in current biosensors, their use is limited to the methods such as fluorescence resonance energy transfer sensors or circular permutation. Here, we describe a first-of-its-kind approach that transforms a protein exhibiting ligand-mediated hinge-bending motion into a highly specific fluorescent biosensor. As a proof-of-concept, a glutamine sensor named Q-SHINE (Glutamine sensor via Split HINgE-motion binding protein) was designed by splitting a glutamine-binding protein into two separate stable domains that, in principle, only dimerize in the presence of glutamine. The application of Q-SHINE to determine glutamine concentrations in solution or mouse serum yielded comparable sensitivity and higher specificity to a conventional glutamine assay kit. Moreover, genetically encoded Q-SHINE could effectively monitor intracellular glutamine levels. Aside from highlighting the reliability of a versatile glutamine sensor, our study opens avenues for researchers who wish to explore the application of ligand-binding proteins as biosensors.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Q-SHINE: a versatile sensor for glutamine measurement via ligand-induced dimerization

Seo

Lim

Jung

et al. 2022

Preprint

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Extracellular chemoreceptor of deca-brominated diphenyl ether and its engineering in the hydrophobic chassis cell for organics biosensing

Chen

Yao

Song

et al. 2022

Chemical Engineering Journal

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Subcellular Localization Defects Characterize Ribose-Binding Mutant Proteins with New Ligand Properties in Escherichia coli

Tavares

Meer

2022

Appl Environ Microbiol

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Periplasmic-binding proteins have been previously proclaimed as a general scaffold to design sensor proteins with new recognition specificities for non-natural compounds. Such proteins can be integrated in bacterial bioreporter chassis with hybrid chemoreceptors to produce a concentration-dependent signal after ligand binding to the sensor cell. However, computationally designed new ligand-binding properties ignore the more general properties of periplasmic binding proteins, such as their periplasmic translocation, dynamic transition of open and closed forms, and interactions with membrane receptors. In order to better understand the roles of such general properties in periplasmic signaling behaviour, we study here the subcellular localization of ribose-binding protein (RbsB) in Escherichia coli in comparison to a recently evolved set of mutants designed to bind 1,3-cyclohexanediol. As proxies for localization we calibrate and deploy C-terminal end mCherry fluorescent protein fusions. Whereas RbsB-mCherry coherently localized to the periplasmic space and accumulated in (periplasmic) polar regions depending on chemoreceptor availability, mutant RbsB-mCherry expression resulted in high fluorescence cell-to-cell variability. This resulted in higher proportions of cells devoid of clear polar foci and of cells with multiple fluorescent foci elsewhere, suggesting poorer translocation, periplasmic autoaggregation and mislocalization. Analysis of RbsB mutants and mutant libraries at different stages of directed evolution suggested overall improvement to more RbsB-wild-type-like characteristics, which was corroborated by structure predictions. Our results show that defects in periplasmic localization of mutant RbsB proteins partly explains their poor sensing performance. Future efforts should be directed to predicting or selecting secondary mutations outside computationally designed binding pockets that take folding, translocation and receptor-interactions into account. Importance Biosensor engineering relies on transcription factors or signaling proteins to provide the actual sensory functions for the target chemicals. Since for many compounds there are no natural sensory proteins, there is a general interest in methods that could unlock routes to obtaining new ligand-binding properties. Bacterial periplasmic-binding proteins (PBPs) form an interesting family of proteins to explore to this purpose, because there is a large natural variety suggesting evolutionary trajectories to bind new ligands. PBPs are conserved and amenable to accurate computational binding pocket predictions. However, studying ribose-binding protein in Escherichia coli we discovered that designed variants have defects in their proper localization in the cell, which can impair appropriate sensor signaling. This indicates that functional sensing capacity of PBPs cannot be obtained solely through computational design of the ligand-binding pocket, but must take other properties of the protein into account, which are currently very difficult to predict.

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Engineering of Sensory Proteins with New Ligand-Binding Capacities

Cited by 3 publications

References 123 publications

Q-SHINE: a versatile sensor for glutamine measurement via ligand-induced dimerization

Q-SHINE: a versatile sensor for glutamine measurement via ligand-induced dimerization

Extracellular chemoreceptor of deca-brominated diphenyl ether and its engineering in the hydrophobic chassis cell for organics biosensing

Subcellular Localization Defects Characterize Ribose-Binding Mutant Proteins with New Ligand Properties in Escherichia coli

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